We examined a small, 5.0-kb plasmid from Mycobacterium fortuitum, designated pAL5000. A restriction map of this plasmid was established. The complete sequence of pAL5000 was cloned in 3 different sites (BamHI, EcoRI, and EcoRV) of pBR322, and in the 3 possible orientations relative to the vector-derived sequence. The pBR322:: pAL5000 hybrids were used to transform the Escherichia coli mutant (minA, minB). Two of the hybrid plasmids (pAL15 and pAL51) coded for the same protein in the minicells, indicating that a sequence of mycobacterial plasmid DNA may function as a promotor recognised by the transcription-translation apparatus of E. coli.